Wallach's Interpretation of Diagnostic Tests: Pathways to Arriving at a Clinical Diagnosis (1398 page)

   Most cultures are incubated at 37°C; cultures from skin or superficial lesions should also be incubated at 30–32°C to improve isolation of mycobacteria that are common pathogens at these sites, such as
M
.
marinum
and
M
.
haemophilum
. Cultures are incubated in 3–10% CO
₂
. Broth media may be monitored on automated platforms, allowing earlier detection time and providing organisms for identification using molecular genetic tests. Clear, agar-based solid media, like Middlebrook media, provide sensitive isolation of
M
.
tuberculosis
, early detection of “microcolonies,” and preliminary, presumptive identification by microcolony morphology. Selective media, which contain a variety of antibiotic agents, may be inoculated for specimens when heavy contamination is likely. Because pathogenic mycobacteria may be inhibited by selective media, nonselective media must always be included. If
M
.
haemophilum
infection is suspected, media supplemented with blood, hemin (X factor strip), or ferric ammonium citrate should be inoculated. Inoculation of parallel cultures, with one culture exposed to light during the early growth phase, and one incubated in the dark only, can be used to determine characteristics of pigment formation.

   Gram and AFB staining is performed on growth from positive AFB cultures; further testing for identification and susceptibility, as appropriate, is performed.

   Growth rate and pigment formation, including photoreactivity, are used to initially characterize non–
M. tuberculosis
mycobacterial species (NMTB) and help to determine the panel of tests required for full identification. Rapidly growing mycobacterial species yield mature colonies within 10 days after subculture.

   Newer technologies for definitive identification of isolates have replaced biochemical and phenotypic testing in many laboratories. The NAP (
p
-nitro-acetylamino-hydroxypropiophenone) test may be used to rapidly identify
M
.
tuberculosis
. Nucleic acid probes are available for identification of
M
.
tuberculosis
complex,
M
.
avium
complex (MAC),
Mycobacterium kansasii
, and
M
.
gordonae
. Nucleic acid sequencing technology is emerging as an important tool for identification of mycobacteria in reference laboratories.

Turnaround Time

   Cultures are incubated for 6–8 weeks. Specimens with a positive AFB smear or direct molecular test result should be incubated for an additional 4 weeks before signing out as negative.

   In positive cultures, several additional weeks may be required for isolation, identification, susceptibility testing, and further characterization, as needed.

   Antimicrobial susceptibility testing should be performed on initial isolates, if
M
.
tuberculosis
, as well as isolates from cultures, positive longer than 3 months after initiation of therapy. Susceptibility testing should also be performed on most NMTB isolates.

   The primary panel for TB isolates include isoniazid, rifampin, ethambutol, and pyrazinamide. For isolates resistant to rifampin or any other two drugs from the primary panel, a secondary panel is performed, including amikacin; capreomycin; cycloserine; ethionamide; kanamycin; PAS; and streptomycin at a low level and a high level.

   Various species-specific drug panels are used for significant NMTB isolates.

   Using optimal growth, identification, and susceptibility testing systems, complete identification and susceptibility testing should be completed for the majority of
M
.
tuberculosis
isolates within 4 weeks of submission of specimen to the lab.